Huntiella moniliformis. These molecular mechanisms are also compared to the underlying mechanisms that control sex in Huntiella omanensis, a closely related, but self-sterile, species. The main finding was that H. omanensis displayed mating-type dependent expression of the a- and α-pheromones. This was in contrast to H. moniliformis where both pheromones were co-expressed during vegetative growth and sexual development. Furthermore, H. moniliformis also expressed the receptors of both pheromones. Consequently, this fungus is likely able to recognize and respond to the endogenously produced pheromones, allowing for self-fertility in the absence of other key mating genes. Overall, these results are concomitant with those reported for other unisexual species, but represent the first detailed study considering the unisexual behaviour of a filamentous fungus.Homothallism (self-fertility) describes a wide variety of sexual strategies that enable a fungus to reproduce in the absence of a mating partner. Unisexual reproduction, a form of homothallism, is a process whereby a fungus can progress through sexual reproduction in the absence of mating genes previously considered essential for self-fertility. In this study, we consider the molecular mechanisms that allow for this unique sexual behaviour in the saprotrophic ascomycete;  MAT1 locus in ascomycetes, harbours genes that represent either the MAT1-1 or the MAT1-2 idiomorph [MAT1-1-1 gene defines the MAT1-1 idiomorph, but can co-exist at this locus with other genes such as MAT1-1-2 and MAT1-1-3. Similarly, the MAT1-2 idiomorph is defined by the MAT1-2-1 gene, though other genes can also be present [Sexual reproduction in fungi commonly requires an interaction between strains that carry different genetic information at the mating type locus . This reMAT1-1 or MAT1-2 idiomorph are termed heterothallic and are self-sterile, other fungi are able to sexually reproduce even in the absence of a partner [MAT1-1-1 and MAT1-2-1 genes in a single cell, as seen in Gibberella zeae [Aspergillus nidulans [MAT idiomorphs and thus undergo sexual reproduction in the absence of genes that are usually considered essential for sex. To date, unisexuality has been described in only five species, including the human pathogens, Cryptococcus neoformans and Candida albicans [While isolates that possess either the  partner . This sella zeae  and Aspenidulans . Other mnidulans , 8. Unisalbicans –13.A. nidulans [Sordaria macrospora [G. zeae [G. zeaeα-factor pheromone has been knocked out and actually enhances facultative outcrossing in female isolates. [S. macrospora where the pheromone receptors have been deleted [Fungi typically depend on protein-based sexual pheromones to attract potential mating partners and as such, pheromones are usually expressed in a mating type-dependent manner , 15. Desnidulans , Sordaricrospora  and G. z[G. zeae . This hasolates.  Likewise deleted . This inCa. albicans and Cr. neoformans [Ca. albicans relies on the inactivation of the Bar1 protease, an enzyme usually required for the proteolytic breakdown of the endogenously produced α-factor pheromone. Upon Bar1 inactivation, the pheromone is not broken down and the cell thus recognizes it as a signal that a compatible mating partner is present. This leads to self-mating of a cells which is dependent on the activation of the α-factor receptor [α cells of Cr. neoformans do not produce endogenous a-factor pheromone, but rely on the presence of a few or distant a cells to produce the pheromone which in turn enhances the fusion of α cells and enables same sex mating [Pheromone expression has been intimately linked to the unisexual abilities of both oformans , 11. Unireceptor . In contHuntiella moniliformis, is a haploid filamentous ascomycete that reproduces unisexually [H. omanensis, are heterothallic and require a partner for mating [Ca. albicans [Cr. neoformans, both of which are yeasts [The primary subject of this study, sexually . This isr mating . Since valbicans , 20–22 ae yeasts , 23–28.H. moniliformis. Using a comparative transcriptomics approach, we sought to identify differences in gene expression between the vegetative and sexual stages of growth in H. omanensis and H. moniliformis. Ultimately, this comparison revealed key gene expression differences between heterothallic and unisexual species that we believe contribute, at least partly, to the unique sexual behaviour seen in H. moniliformis.The main objective of this study was to elucidate the underlying molecular pathway associated with unisexual reproduction in H. moniliformis and H. omanensis previously used in the study of Wilsonet al. [H. moniliformis, two distinct forms of isolate CMW36919 (CBS 144008) were used. One of these was sexually-competent, producing abundant ascomata and ascospores, while the other maintained a strictly vegetative growth form. For H. omanensis, three different isolates were used. Isolate CMW11056 (CBS 118113) is a mixed mating-type culture (MAT1xMAT2) representing the sexually reproducing isolate type. The vegetative isolates , represent MAT1 and MAT2 isolates, respectively. The two vegetative cultures were isolated as single ascospore progeny from the mixed mating-type culture CMW11056 [Isolates of net al.  were use CBS 1181 is a mix-1 malt extract  and 20 g.L-1 agar  supplemented with thiamine hydrochloride  and streptomycin sulphate salt . These are forthwith referred to as MEA-ST plates. The cultures were maintained at room temperature for the duration of the study. All cultures used in this study are preserved in the culture collection (CMW) of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa and at the Westerdijk Fungal Biodiversity Institute, The Netherlands.Cultures were grown and maintained on 2% malt extract agar plates  and H. omanensis  cultures.Total RNA was extracted from 5–7 day-old cultures. This time period ensured that cultures did not reach the stationery phase, but gave enough time for sexual reproduction to take place. Nine MEA-ST plates were used per culture, allowing for three biological and three technical replicates. Mycelium was harvested from the vegetatively growing ® Mini Kit  following the manufacturer’s protocols with the following modifications: the RLC buffer was used as the extraction buffer and the optional on-column DNase 1 digestion was included. The integrity of the total RNA was assessed by 2% (w/v) agarose gel electrophoresis at 120V for 25 min and the concentration was estimated using an ND 1000 Spectrophotometer . The RNA was further subjected to quality testing using the Experion™ automated electrophoresis system  at the ACGT Microarray Facility . Dynabead®-based mRNA enrichment , cDNA synthesis and library preparation were performed at the Central Analytical Facilities (CAF) at the University of Stellenbosch (South Africa). The RNA sequencing was completed at the same facility using the Ion Proton Platform and PI™ Chip system.The harvested tissue was flash frozen in liquid nitrogen and ground to a fine powder using a mortar and pestle. RNA extractions were performed using the RNeasyCLC Genomics Workbench V7.5  was used to filter out raw reads with a Phred quality score of below 20 (Q ≤ 0.01) and a read length of more than 300 bp. Up to two terminal ambiguous nucleotides were trimmed from the remaining reads. To remove any reads representing the rRNA fraction of the total RNA, the filtered and/or trimmed reads were mapped to a species-specific contig harbouring only the rRNA sequences using the RNA Seq (Legacy) tool in CLC Genomics Workbench. To ensure optimal mapping, a minimum length fraction of 0.5 and a minimum similarity fraction of 0.8 were used. Reads that had not mapped to the rRNA contigs were then used in further analyses.H. moniliformis  and H. 0000000, ) using We models , 32. Thei et al. .Reads Per Kilobase of exon model per Million mapped reads) corrects for differences in expression levels between genes of different lengths and allows for accurate intra- and inter-sample comparisons [For the accurate comparisons of gene expression across samples, a two-step normalization of the expression values was conducted. Firstly, RPKM values were used to represent relative expression. RPKM  and used as queries in local BLASTn and tBLASTx searches against the draft genome assemblies using CLC Genomics Workbench.Genes known to be involved in sexual reproduction in other ascomycete fungi were investigated for their presence and possible DE in the two  species . These i species , as wellrevisiae , 39 and  species . The a- proteins  were alsH. moniliformis samples and an average of ~21 million reads per library for the H. omanensis samples  was extracted from each of the cultures used in this study . Sequenc samples . The rawanalyses .H. moniliformis and H. omanensis genomes  were expressed across both isolate types. In the 31 Mb genome of H. omanensis, 7 923 (94%) of the total 8 394 predicted genes were expressed across the three isolate types. A gene was considered expressed if three or more unique gene reads mapped to its annotation [Of the predicted 6 864 genes in the 25 Mb notation  and a nonotation . The majH. moniliformis isolate, 156 were highly expressed  were up-regulated in the sexually reproducing culture, while MAT1-1-2 showed no evidence of differential expression. MAT1-2-7 also showed a general pattern of up-regulation during sexual reproduction, but high intra-sample variation rendered this result insignificant. This gene was discovered using bioinformatics analyses in a previous study [MAT1-2 idiomorphs in a variety of other ascomycetes, this gene was named MAT1-2-7. This gene has recently been reported in Knoxdaviesia species [MAT locus in H. omanensis and the increase in its expression during sexual reproduction suggests that it has some unknown function during sexual reproduction. The H. moniliformis MAT1-2-1 gene did not show a significant increase in expression during sexual reproduction. The truncated H. moniliformis MAT1-2-7 gene was not expressed in either the vegetative or sexually reproducing cultures. This gene possesses a premature stop codon in H. moniliformis and it has been proposed that it would produce a non-functional gene product of only 48 aa in length [The  species  were ideus study  and has  species . Its assn length .Huntiella species  and its receptor (down-regulated). Again, H. moniliformis exhibited a different pattern and while the a-factor pheromone showed a decrease in expression in the sexually reproducing culture, the expression of the α-factor pheromone increased by almost 20-fold (p<0.001). Additionally, the mpkB, ste20 and ste23 genes  were also up-regulated in the sexually reproducing H. moniliformis culture.The only genes that were differentially expressed in this pathway in H. moniliformis to similar changes associated with heterothallic mating in a closely related species. The results showed that there are significant differences in the pheromone production in these two species.Unisexual reproduction enables the production of sexual spores in fungi that usually require an opposite mating partner. To date, unisexuality has been observed in only five species; four ascomycetes and one basidiomycete. Although three of these species are filamentous fungi, the underlying molecular mechanisms enabling this form of self-fertility have only been characterized in the two yeast species. We compared the gene expression changes associated with unisexual reproduction in the filamentous ascomycete H. omanensis were very similar to the general expression profiles in self-sterile model fungi such as N. crassa. Although individual isolates of N. crassa possess genes for both the α- and a-factor pheromones, they are expressed in a mating-type specific manner [α-factor pheromone, while mat a (= MAT2) individuals express only the a-factor pheromone. Sexual reproduction is then initiated only in the presence of a mating partner of the opposite mating-type, despite the fact that both partners express both pheromone receptor genes [H. omanensis also follows this pattern. Interestingly, there was also some evidence of mating-type dependent receptor expression, a profile not observed in N. crassa or other heterothallic species [A comparative transcriptomics approach revealed that changes in the expression of the pheromone precursor genes in the heterothallic c manner . Thus, mor genes . Our res species .H. moniliformis provides a putative molecular mechanism that enables the species to reproduce unisexually. These genes exhibit neither the same mating-type dependent expression nor the differential expression seen in H. omanensis. However, they do display expression profiles similar to other homothallic species such as S. macrospora [S. macrospora, possess both MAT1-1 and MAT1-2 genes, and thus, despite these genes being under mating-type control, both pheromones can be expressed. In contrast, H. moniliformis is able to achieve this expression profile, while harbouring only the MAT1-2-1 gene.The expression pattern of the pheromones in crospora  and thusH. omanensis, the α-factor receptor showed down-regulation in the sexually reproducing isolate, while the H. omanensisa-factor receptor as well as both receptors in H. moniliformis showed no evidence of differential expression. This result indicates that differential expression of the receptors is not important to sexual reproduction in these species and that constitutive expression is sufficient. This could also indicate that differential expression of the pheromones themselves controls the activation of sexual reproduction, irrespective of whether it is a heterothallic or homothallic interaction.Pheromone expression is only part of the sexual activation pathway. This is because pheromones can only elicit their intended response if recognized by the appropriate pheromone receptor. In our study, both receptors were expressed at appreciable levels by both fungal species studied. This indicates that the pheromone response pathway is being utilized. In MAT1-2 idiomorph-associated gene, MAT1-2-7. This gene was previously detected using de novo gene prediction software [H. omanensis. Its position upstream of the archetypical MAT1-2-1 gene in MAT2 isolates coupled with its absence in the MAT1 isolates highlighted the role that it might play in mating. In the present study, MAT1-2-7 had no detectable expression level in vegetatively growing isolates, but was shown to be expressed in sexually reproducing cultures. This expression pattern, in addition to its idiomorphic position, strongly supports a role for the MAT1-2-7 gene in sexual reproduction. Because it is truncated in H. moniliformis, we believe it is likely that this gene plays a role in the inhibition of self-fertility.The results of this study have also provided the first experimental validation for the expression of the software  and is tH. omanensis was included in this study because it represents a typical heterothallic species. It was consequently able to provide a basis for comparison of gene expression changes that occur during a sexual interaction in Huntiella species. This was especially important due to the unavailability of MAT1 H. moniliformis isolates. Thus, following the model proposed for H. omanensis, expression of the a-factor pheromone in H. moniliformis is either directly or indirectly influenced by genes present at the MAT1-2 idiomorph. This same model would suggest that the α-factor pheromone should be controlled by genes at the MAT1-1 idiomorph. However, H. moniliformis MAT2 individuals also express this gene. Taken collectively, these results suggest that MAT2 individuals of H. moniliformis have been able to overcome the mating-type dependent expression of the pheromones that would otherwise prevent homothallic behaviour in typically heterothallic species  are not only required for the activation of mat+ functions, but also for the repression of mat- (= MAT1-1) fertilization functions. Thus, when there is a mutation in the mat+ gene, FPR1, the repression of fertilization is lifted and the mutant is rendered self-fertile. We suggest that this could be similar to what occurs in H. omanensis, where a full length MAT1-2-7 gene represses the α-factor pheromone, rendering the MAT2 isolates self-sterile. However, in H. moniliformis, the MAT1-2-7 gene is truncated, the repression of the α-factor pheromone does not occur and mating-type independent expression is possible. This in turn leads to self-activation, self-fertility and culminates in unisexual reproduction.The hypothesis that the truncated anserina . Here, da- and α-factor pheromones is responsible for the unique ability of the MAT2 individuals of H. moniliformis to complete a sexual cycle in the absence of a MAT1 mating partner. As presented above, the role of pheromone expression in unisexuality has been previously investigated in both Ca. albicans and Cr. neoformans [Ca. albicans, and we hypothesize that endogenous production of both the a- and α-factor pheromones by a single MAT2 individual is sufficient to initiate the sexual cycle in H. moniliformis. We believe that this is the most probable explanation for the data presented here but acknowledge that other plausible hypotheses may also exist.The results of this study suggest that the mating-type independent expression of the oformans , 23–26. H. moniliformis was originally assumed to be a sexual process. This was partly due to the similarity of the reproductive structures and spores found in H. moniliformis and the heterothallic H. omanensis [H. moniliformis further substantiates this assumption. However, it is worth noting that another spore-producing process, known as haploid fruiting, also enables spore production in single mating type isolates. Although similar to unisexuality in that way, this process is not necessarily linked to sexual reproduction or the formation of a diploid state [Schizophyllum commune when the mycelium is physically damaged [H. moniliformis produces progeny, the currently available data supports a sexual process.The unisexual pathway exhibited by manensis . The appid state –56. It mid state . Haploid damaged  or when  damaged . While tMAT1-2-7 gene in order to enhance our understanding of the role these genes play in unisexuality. Furthermore, studies utilizing synthetic pheromones will be conducted in an attempt to initiate unisexual behaviour in H. omanensis.This study utilized a transcriptomics approach that has some inherent limitations. The most important is that conclusions drawn from the data assume that gene expression is directly correlated with the production of a functional protein product. In addition, pheromones are often expressed as pre-pheromone proteins that require post-translational modification. Therefore, future studies will include the functional characterization of the two pheromone genes as well as the S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.S8 Table(XLSX)Click here for additional data file.S9 Table(XLSX)Click here for additional data file.S10 Table(XLSX)Click here for additional data file.


Objective. This study explored the mechanism of herb-partitioned moxibustion (HM) on dextran sulfate sodium- (DSS-) induced ulcerative colitis (UC) from the miRNA perspective. Methods. Rats were randomly divided into 3 groups . The UC and UCHM groups were administered 4% DSS for 7 days. The UCHM group received HM at the Tianshu . The effect of HM on UC was observed and the miRNA expression profile in the colon tissues was analyzed. Results. Compared with the UC group, the body weights were significantly higher in the UCHM group on day 14 (P < 0.001); the macroscopic colon injury scores and microscopic histopathology scores in the UCHM group decreased (P < 0.05); and there were 15 differentially expressed miRNAs in the UCHM group. The changes in miR-184 and miR-490-5p expression levels on the UC were reversed by HM intervention. Validation using qRT-PCR showed that two miRNAs expression trend was consistent with the sequencing results. Conclusion. HM at ST25 might regulate miR-184 and miR-490-5p expression, act on the transcription of their target genes to regulate inflammatory signaling pathways, and attenuate inflammation and tissue injury in the colons of rats with DSS-induced UC. Ulcerative colitis (UC) is a group of chronic, nonspecific, intestinal inflammatory diseases with unclear etiology. The incidence of UC gradually increases annually in response to changes in lifestyle, dietary structure, and living conditions , 2. AccoRecently, the association between noncoding RNAs and inflammation has been closely investigated. MicroRNAs (miRNAs) are small noncoding single-stranded RNAs that are 18–25 nucleotides in length. They regulate gene expression through binding to the 3′-untranslated region (UTR) of mRNAs encoding target proteins to play an epigenetic posttranscriptional role. The biological functions of miRNAs are associated with adaption to physiological and pathological environments and the reversal or change of gene expression in completely differentiated tissues . miRNAs Joos et al. performed a randomized controlled study to show that acupuncture and moxibustion of traditional Chinese medicine (TCM) could improve the quality of life of UC patients . Over thThe NanoDrop-2000 spectrophotometer was supplied by NanoDrop Technologies . The TRIzol reagent was supplied by Invitrogen . The TapeStation 2200 , Illumina TruSeq™ Small RNA Sample Prep Kit , Qubit® 2.0 Fluorometer using the dsDNA HS and/or BR assay kit , Illumina HiSeq 2000 sequencer , 36–50 kDa DSS , Bio-Rad CFX96TM Real-Time PCR system , TruSeq PE Cluster Kit v3-cBot-HS , RNase inhibitor , and iQTM SYBR® Green Supermix  were obtained from the indicated manufacturers.Healthy adult male Sprague-Dawley (SD) rats with body weights of 180 ± 20 g were provided by the Experimental Animal Center of Shanghai University of Traditional Chinese Medicine and were purchased from Vital River Laboratory Animal Technology Co. Ltd. . The license for the use of experimental animals was SCXK (Beijing) 2012-0001. The housing conditions were clean-grade, 12 h of alternating day and night (illumination from 7 a.m.–7 p.m.), 20 ± 2°C room temperature, and 50–70% indoor humidity. After 1 week of adaptive feeding, the formal experiments began. The experiments were performed in strict accordance with the guidelines for experimental animals of the National Institute of Health (NIH) of the USA. Animal treatment during the experimental process followed the regulations of “Instructive Notions with respect to Caring for Laboratory Animals” released by the Ministry of Science and Technology, China.After 1 week of adaptive feeding, the formal experiments began. The 36 rats were randomly divided into the following 3 groups: normal control (NC) group, UC model (UC) group, and herb-partitioned moxibustion (UCHM) group. Each group had 12 animals. The UC and UCHM groups both received 4% DSS in water ad libitum for 7 days. After model establishment, two animals from each group were selected for hematoxylin and eosin (HE) staining to determine whether the model was successfully established. After the model was successfully established, the animals were provided 1% DSS in water ad libitum for 7 days. The UCHM group was also provided with the moxibustion intervention. The body weights of the rats in each group were measured on the first day of the intervention .Coptis chinensis, Radix aconiti lateralis, Cortex Cinnamomi, Radix aucklandiae, Flos carthami, Salvia miltiorrhiza, and Angelica sinensis) mixed and stirred with yellow wine to form a thick paste. The medicinal cake was prepared with 1 cm and 0.45 thicknesses using a mold. The moxa cone was prepared with a 0.6 cm diameter and height using a mold; the weight was 90 mg. The rats were immobilized using a fixator, and the ST25 points  were exposed. Rat hair at the ST25 points was shaved one day in advance. The prepared moxa cone was placed on the top of a medicinal cake, and the medicinal cake was placed at the ST25 points . The moxa floss was lit; each acupoint used 2 moxa cones per time point. Moxibustion was performed once every day in the morning for intervention for 7 days  points were selected. The medicinal cake was Chinese medicine powder . The distal colon at a length of 6–8 cm was collected 1 cm from the anus. Each colon was divided into 2 parts. One part was fixed in 4% paraformaldehyde. The other part was minced and placed in a cryotube, frozen in liquid nitrogen for 1 h, and stored in a −80°C freezer for future use.The collected distal colon was cut open along the mesenterium and spread flat. Adhesion and bleeding of the mucosa were observed. The scoring of the distal colon of the rats was performed according to the Score Criteria of Colonic General Damage , 21.μm thin sections. Histopathological changes of the colon were observed using HE staining under a light microscope. Microscopic scoring of the colon was performed according to the Score Criteria of Colonic Histological Damage [Colon tissues fixed in paraformaldehyde for 24 h were removed, cleaned, trimmed, dehydrated, embedded, and sectioned into 4 l Damage .μg of total RNA from each sample was used to construct the small RNA library. Different index tags were selected for library construction based on the operational manual of the TruSeq Small RNA Sample Prep Kit . The quality of the library after construction was examined using the Agilent 2200.Two colon tissues were collected from each group. Total RNA was extracted from the colon tissues using the TRIzol method. The purity and concentration were detected using the NanoDrop 2000. The RNA integrity was detected using the Agilent 2200. Total RNA samples with RIN (RNA integrity number) values higher than 8 were used for library construction. A total of 3 P values were calculated. FC ≥ 1.5 was set as upregulation and ≤0.5 as downregulation .This experiment used the TruSeq PE Cluster Kit v3-cBot-HS (Illumina) reagent to generate clusters on cBOT. Next, the single-terminal sequencing program was performed on the HiSeq 2000 sequencing platform. The data collection software provided by Illumina was used to control the sequencing process and real-time data analysis. The results were compared with the reference sequences, and DEseq was used for the analysis. The fold changes and http://www.microrna.org/microrna/home.do), miRDB (http://mirdb.org/miRDB/), and TargetScan6.2 (http://www.targetscan.org/vert_60/).The miRNA target gene prediction was performed using a combination of MIRanda  enrichment analysis was performed, with a P value < 0.05 indicating significant enrichment.Signaling pathways with significant enrichment of target genes were analyzed using −ΔΔCt method was performed to analyze relative miRNA expression in 5 replicate samples. The one-way analysis of variance (ANOVA) method was performed to assess the significance of differences in the relative miRNA expression levels between the 3 groups (P < 0.05).The samples were amplified, and 2 miRNAs (miR-184 and miR-490-5p) were validated using quantitative real-time PCR. U6, miR-184, and miR-490-5p were synthesized and purified by RiboBio . The 225, P75). If the data conformed to the normal distribution, the comparison of differences among groups was performed using one-way ANOVA. If the variances were homogeneous, the pairwise comparison was performed using the least significant difference (LSD) test. If the variances of all groups were not homogenous, the nonparametric Kruskal-Wallis H test was performed for the analysis. The pairwise comparison was performed using the Nemenyi method. If the data did not conform to the normal distribution, the rank sum test was performed. The significance level of the statistical examination was α = 0.05. P < 0.05 indicated that the difference had statistical significance.The SPSS 22.0 software was used for the analysis. The normality test and the homogeneity of variance test were performed on the data. The data were presented as the mean ± standard deviation P < 0.05; except for day 7) and significantly decreased on days 10–14 (P < 0.001) compared to the NC group rats. The body weights of rats in the UCHM group increased on days 11-12 (P < 0.05) and significantly increased on days 13-14 (P < 0.001) compared to the UC group rats (The body weights of the rats with DSS-induced UC significantly changed. The body weights of rats in the UC group decreased on days 4–8 (oup rats .P < 0.05). The macroscopic colon injury scores in the UCHM group were significantly decreased compared to the scores in the UC group (P < 0.05)  .P < 0.05). Conversely, the microscopic scores of colon tissues in the UCHM group were decreased compared to the rats in the UC group (P < 0.05)  .Through the above observations of body weight, gross colon tissue injury, and histopathology, we found that HM had better intervention effects on DSS-induced UC and could correct histopathological changes of the colon, attenuate intestinal inflammation, and increase the body weight of the rats. Next, colon tissues were collected from two rats per group for total RNA extraction. Small RNA sequencing technology was used to observe the effect of HM on the miRNA expression profiles in the colon tissues of rats with DSS-induced UC.P values smaller than 0.05 indicated significant differences. Expression values lower than 0.001 were filtered. The heat map of the differentially expressed miRNAs was analyzed using the Cluster 3.0 and Treeview software to intuitively assess the homogeneity in each sample and the expression differences among all of the groups. The heat map showed that the expression of two samples in each group was more consistent and that there were significant differences between two groups. There were 49 differentially expressed miRNAs in the colon tissues of the UC group compared to the NC group, of which 47 were upregulated and 2 were downregulated; miR-184 and miR-18a-5p were both downregulated . The target genes of rat miRNAs can be predicted using 3 databases . miRNA-184 had 1130 target genes. The relevant top 30 pathways that were closely associated with the occurrence and development of intestinal inflammation and malignant tendencies in UC included the TNF signaling pathway, Ras signaling pathway, pathway in cancer, NF- pathway . miR-490 pathway .P < 0.05). The miR-184 expression level increased and the miR-490-5p expression level decreased in the UCHM group compared to the UC group (both P < 0.05) (Figures The expression of the 2 miRNAs that were reversed by HM was validated using the qRT-PCR method. The miR-184 expression level decreased and the miR-490-5p expression level increased in the UC group compared to the NC group (both  Figures .The major symptoms of UC are abdominal pain, diarrhea, and bloody stool. The active period and the remission period usually alternate, and the disease course is very long. Currently, UC is considered to be associated with a variety of factors, including genetics, environment, diet, intestinal flora, and immunity . HoweverκB to regulate the NF-κB signaling pathway [κB and STAT3 could be regulated to influence the IL-6 expression level. In contrast, miR-214 inhibitors reduced colitis in UC patients and tissues with DSS-induced colitis [With the development of microarrays and high-throughput sequencing, UC disease-associated miRNAs are gradually being elucidated. These miRNAs can regulate inflammatory cell chemotaxis, transcription factor activity, intestinal epithelium barrier functions, and autophagy activity –36. We h pathway . In colo colitis . miR-532 colitis . miR-532 colitis . miR-340 colitis . FinallyThe inflammatory reaction present in local tissues after infection and injury in the body is a common and important basic pathological process. miRNAs play important roles in immune regulation in the colon. Acupuncture and moxibustion have immune regulatory and inflammation inhibition functions, and moxibustion can inhibit colitis in UC subjects –45. The κB signaling pathway, miRNAs in cancer, colorectal cancer, and AMPK signaling pathway. Similarly, the miR-490-5p target genes were involved in pathways associated with UC, including the cGMP-PKG signaling pathway, cAMP signaling pathway, neurotrophin signaling pathway, HIF-1 signaling pathway, and endocrine and other factor-regulated calcium reabsorption. HM regulated small RNA expression. Thus, HM may act on the transcription of their target genes, thereby attenuating intestinal inflammation in rats with DSS-induced UC. In the future, in-depth studies on the 2 small RNAs will be performed to investigate the mechanism of action of HM at the ST25 points on DSS-induced UC.Target gene predictions for miR-184 and miR-490-5p and the pathway analysis of the target genes of these 2 small RNAs showed that potential target genes of miR-184 were involved in some pathways that were closely associated with UC, such as the TNF signaling pathway, Ras signaling pathway, pathway in cancer, NF-


Abiraterone and enzalutamide are currently approved for mCRPC patients. Both drugs have distinct mechanisms of action and may have different toxicity profile. There are limited data comparing the side effects of abiraterone and enzalutamide. We performed a meta-analysis of randomized controlled trials (RCT) to better characterize the risk of adverse events associated with both drugs.We performed a literature search on MEDLINE for studies reporting abiraterone and enzalutamide side effects from January 1966 to July 31, 2015. Abstracts presented at ASCO meetings from 2004 to 2015 were selected manually. Phase III RCT were included in analysis. We assessed the risk of adverse events reported in RCT by performing two meta-analyses: abiraterone-prednisone vs. placebo-prednisone  and enzalutamide vs. placebo . Summary of incidence, relative-risks (RR), and 95% confidence intervals (CI) were calculated using random-effects or fixed-effects models based on the heterogeneity of included studies.Overall, enzalutamide was not associated with all-grade (RR 1.06 - 95% CI 0.67-1.65) or grade ≥3 (RR 0.81 - 95% CI 0.28-2.33) cardiovascular events, but was associated with increased risk of all-grade fatigue (RR 1.29 - 95% CI 1.15-1.44). On the other hand, abiraterone was associated with increased risk of all-grade (RR 1.28 - 95% CI 1.06-1.55) and grade ≥3 (RR 1.76 - 95% CI 1.12-2.75) cardiovascular events, but was not associated with all-grade (RR 0.85 - 95% CI 0.58-1.23) or grade ≥3 (RR 1.07 - 95% CI 0.97-1.19) fatigue.In this meta-analysis, abiraterone was associated with an increased risk of cardiovascular events, while enzalutamide was associated with an increased risk of fatigue. Prostate cancer is the second leading cause of cancer death among men . AndrogeOverall, abiraterone and enzalutamide have a favorable toxicity profile. The most frequent adverse events are hot flashes and fatigue for enzalutamide and fluid retention, transaminase increases and hypokalaemia for abiraterone. This topic is of particular relevance in patients with mCRPC, who may have a high number of comorbidities.In this report, using a meta-analysis design, we sought to investigate the differential incidence of cardiovascular events and fatigue as side effects of abiraterone and enzalutamide in mCRPC patients. The two most important clinical toxicities reported for these drugs are cardiotoxicity for abiraterone and fatigue for enzalutamide. Other exploratory toxicities were not included to avoid the risk of positive findings due to multiple comparisons.Figure p = 0.01) and in a 28% percent increase in all-grade cardiac disorder adverse event . On the other hand, abiraterone plus prednisone was neither associated with an increased risk of all-grade nor high-grade fatigue. These results are presented in Figures Abiratrone was used in association with prednisone and compared to placebo plus prednisone. Abiraterone plus prednisone was associated with a statistically significant 76% increase in the risk of high-grade cardiac disorder adverse events , but not the risk of high-grade fatigue . Figures Enzalutamide was not associated with an increase in the risk of all-grade or high-grade cardiac disorder adverse event. However, it was associated with an increased risk of all-grade fatigue by 29%  of these drugs and reported that their use is associated with an increased risk of all-grade cardiotoxicity  but not with an increased risk of grade ≥3 cardiac events, compared with a placebo. However, the analyzed agents included the novel CYP-17 inhibitor TAK 700 which, due to the negative results in terms of OS achieved, is not approved for the treatment of prostate cancer  and andn = 6e cancer . Adminisbidities . However disease  15]..n = 6) oTherefore, we suggest careful consideration of the risks and possible benefits of abiraterone therapy for mCRPC patients with cardiovascular comorbidities  or a history of cardiac disease. Lastly, it may be reasonable to recommend performing a detailed cardiovascular assessment prior to starting Abiraterone and periodical monitoring during treatment.As much as abiraterone-related cardiotoxicity can represent a life-threatening side effect, enzalutamide-related fatigue should not be disregarded. Even if this analysis does not show a higher risk of grade ≥3 fatigue for patients treated with Enzalutamide, our results demonstrate an association with increased risk of all-grade fatigue. It is commonly acknowledged that fatigue may have a major impact on patients’ quality of life , self-caAs both abiraterone and enzalutamide demonstrated similar efficacy prior to and after chemotherapy in mCRPC patients  4], the, the4], We are aware that several limitations apply to this study. First, our analysis hinges on solely 4 RCT and not on individual patient data; second, the analyzed RCT adopted different versions of the Common Terminology Criteria for Adverse Events (CTCAE); third, our findings are based on RCT data which might not reflect the real-world setting due to the RCT strict inclusion/exclusion criteria; fourth, other exploratory toxicities were not included.Despite these drawbacks, our analysis shows an increased risk of all-grade and grade ≥3 cardiovascular events for abiraterone and a higher risk of all-grade fatigue for enzalutamide. These findings provide a better understanding of the divergent side effect profile of these drugs and may help selecting the more appropriate therapy for each patient on the basis of his baseline comorbidities, cardiovascular history, and conditions.MEDLINE was searched from January 1966 to 31 May, 2016 and ASCO meetings abstracts were manually selected from 2004 to 2016. The MeSH terms used for searching PubMed and the Cochrane Library were ‘abiraterone’ or ‘enzalutamide’. The ASCO University abstracts were searched using the name of the drugs and the terms ‘phase II’ or ‘phase III’. Search was restricted to randomized phase II or III clinical trials reported in English. Title and abstracts of all the original reports identified were reviewed by two independent authors (R.B.M. and F.A.S) for inclusion in this meta-analysis. Cases of disagreement were adjudicated by consensus among reviewers. The full texts of all potentially relevant studies were downloaded and reviwed by two independent authors (R.B.M. and F.A.S) for inclusion in this meta-analysis. Cases of disagreement were adjudicated by consensus among reviewers.The inclusion criteria for this meta-analysis were:  prospectIdentified abstracts were then collected and coded by two investigators (R.B.M. and F.A.S) according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement  and any The outcomes included in this meta-analysis of treatment toxicities were all grade and high-grade any cardiac event and fatigue. Cardiac toxicity was defined by the following events: ischaemic heart disease, myocardial infarction, supraventricular tachyarrhythmias, ventricular tachyarrhythmias, cardiac failure, and possible arrhythmia-related investigations, signs, and symptoms. Treatment effects were pooled as relative risks (RR). All comparisons were based on two-tailed tests and p-values lower than 0.05 were considered significant.2. Heterogeneity was considered significant whenever the Cochrane Q test yields a p-value under 0.10 or the I2 resulted higher than 50%. If heterogeneity was deemed not to be significant, the generic inverse variance fixed effect (Mantel Haenszel) was used; while the use of the random-effect method (DerSimonian) was reserved for cases of significant heterogeneity. Statistical analysis were carried out using the version 5.3 of the software RevMan.Heterogeneity was accessed using Cochrane Q test and I


To develop and evaluate serological methods for chikungunya diagnosis and research in Nicaragua.Two IgM ELISA capture systems (MAC-ELISA) for diagnosis of acute chikungunya virus (CHIKV) infections, and two Inhibition ELISA Methods (IEM) to measure total antibodies against CHIKV were developed using monoclonal antibodies (mAbs) and hyperimmune serum at the National Virology Laboratory of Nicaragua in 2014–2015. The sensitivity, specificity, predictive values, and agreement of the MAC-ELISAs were obtained by comparing the results of 198 samples  with the Centers for Disease Control and Prevention’s IgM ELISA . For clinical evaluation of the four serological techniques, 260 paired acute and convalescent phase serum samples of suspected chikungunya cases were used.All four assays were standardized by determining the optimal concentrations of the different reagents. Processing times were substantially reduced compared to the CDC-MAC-ELISA. For the MAC-ELISA systems, a sensitivity of 96.6% and 97.4%, and a specificity of 98.8% and 91.5% were obtained using mAb and hyperimmune serum, respectively, compared with the CDC method. Clinical evaluation of the four serological techniques versus the CDC real-time RT-PCR assay resulted in a sensitivity of 95.7% and a specificity of 88.8%–95.9%.Two MAC-ELISA and two IEM systems were standardized, demonstrating very good quality for chikungunya diagnosis and research demands. This will achieve more efficient epidemiological surveillance in Nicaragua, the first country in Central America to produce its own reagents for serological diagnosis of CHIKV. The methods evaluated here can be applied in other countries and will contribute to sustainable diagnostic systems to combat the disease. Alphavirus genus of the Togaviridae family. The 60 nm – 70 nm diameter virion has a phospholipid envelope and a singlestranded positive-sense ribonucleic acid (RNA) genome that encodes three structural and seven nonstructural proteins  is a reemerging arbovirus that belongs to the proteins , 2. It hproteins . CHIKV imosquito . The dismosquito  – 3. ChiIn December 2013, the Pan American Health Organization (PAHO) reported the first case of CHIKV transmission in the Americas. The virus quickly spread from St. Martin to other countries in the Caribbean, as well as to Central and South America  – 10. ThIn Nicaragua, epidemiological surveillance of chikungunya was based on Immunoglobulin M (IgM) detection in suspected cases after the 4th day of illness . InitialAdditionally, a goal was set to implement a surveillance system similar to the one in place for dengue surveillance, where most of the reagents for dengue-specific IgM antibody detection are produced at the National Virology Laboratory  and distributed to 12 laboratories throughout the country. Therefore, the researchers produced a rabbit hyperimmune serum in order to establish a sustainable chikungunya diagnostic and surveillance system through the development of an additional MAC-ELISA. Furthermore, to help predict epidemiological behavior in the coming years, it was necessary to perform focal and national seroprevalence studies to determine the extent of the current epidemic.To this end, two Inhibition ELISA methods (IEM), using the mAbs or hyperimmune serum, were developed to determine total anti-CHIKV antibodies. The IEMs were based on the methods described by Vazquez and Fernandez for dengue , 16. AllTo determine the cutoff (or positivity threshold) values of both MAC-ELISA methods, 137 samples  were processed. The sensitivity, specificity, predictive values, and agreement of both MAC-ELISA systems in comparison with the CDC-MAC-ELISA were determined using 198 additional samples (116 positive and 82 negative), collected an average of 5.9 days after symptom onset (interquartile range [IQR]: 1). All samples consisted of sera from suspected chikungunya cases collected through the national surveillance programs of Nicaragua. The detailed protocol of the CDC-MAC-ELISA is described elsewhere , 18.These studies were approved by the Institutional Review Boards of the Centro Nacional de Diagnóstico y Referencia (CNDR) of the Ministry of Health of Nicaragua and the University of California–Berkeley . The reference method for the clinical evaluation was the CHIKV real-time Reverse-Transcription-Polymerase Chain Reaction (rRT-PCR) recommended by the CDC.In order to obtain hyperimmune serum (HIS), the vaccine strain CHIKV 181/25 , 14 was Anti-CHIKV gamma globulin was precipitated from the HIS using saturated ammonium sulfate, and then conjugated to horseradish peroxidase (HRP) using the periodate oxidation method . An antiAedes albopictus C6/36 HT cells at 33°C in 175-cm2 flasks for 5 days under Biosafety Level (BSL) 2 conditions since 181/25 is a vaccine strain. Three freeze-thaw cycles were performed, then the flask content was centrifuged at 3 500 rpm for 30 minutes (min), and the supernatant was concentrated by osmosis with refined sugar for 24 hours (hr). The presence of the virus was confirmed by rRT-PCR. The antigen was inactivated with formaldehyde (1 : 2 000) and stored at –80° C until further use.CHIKV antigen was obtained by inoculating the vaccine strain CHIKV 181/25 , 14 intoStrips of polystyrene microwells  were coated overnight at room temperature with 100 µl per well of anti-human IgM immunoglobulin at a concentration of 5 µg/ml and 2.5 µg/ml in carbonate buffer  for the IgM capture ELISA using hyperimmune serum (MAC-ELISA-HIS) and monoclonal antibody (MAC-ELISA-mAb), respectively. Wells were washed three times with phosphate-buffered saline (PBS) containing 0.05% Tween20 (PBS-T) and blocked with 1% bovine serum albumin (BSA) in PBS-T for 30 min at 37°C. Then 50 µl of serum diluted 1 : 20 in PBS-T was added and incubated for 1 hour at 37°C. After four washes with PBS-T, 50 µl of CHIKV antigen (1:25 dilution for MAC-ELISA-HIS and 1:50 for the MAC-ELISA-mAb) was added and incubated for 1 hr at 37°C, then washed four times with PBS-T. HRP-conjugated anti-CHIKV antibodies were then added as follows: for the MAC-ELISA-HIS, 50 µl of a 1 : 10 000 dilution of HRP-conjugated anti-CHIKV HIS for 30 min at 37°C; and for the MAC-ELISA-mAb, 50 µl of a 1:10 000 dilution of HRP-conjugated CHIKV mAb 152 for 1 h at 37°C. After five washes with PBS-T, 50 µl of substrate tetramethylbenzidine  was added for 10 min at room temperature, and the reaction was terminated by the addition of 50 µl of 12.5% sulfuric acid. The optical density (OD) at 450 nm was measured in an ELISA reader . Samples with an OD higher than the average of the negative controls plus 3 standard deviations for MAC-ELISA-HIS and greater than the average of the negative controls plus 4 standard deviations for MAC-ELISA- mAb were considered positive. Positive and negative controls consisted of serum samples with known anti-CHIKV IgM responses, as determined using the CDC-MAC-ELISA, that tested negative for Hepatitis B, C, and human immunodeficiency virus (HIV).In all, 96 well-polystyrene plates (Nunc Immuno MaxiSorp) were coated overnight at room temperature with 100 µl per well of 10 µg/ml anti-CHIKV HIS (IEM-HIS) or with 5 µg/ml of CHIKV mAb 187  in carbonate buffer. The plates were washed three times with PBS-T and blocked with 1% BSA in PBS-T for 30 min at 37°C. Then, 100 µl of antigen (1 : 100 dilution for both IEM) was added for 1 hr at 37°C. After four washes with PBS-T, 100 µl of serum , positive control (1:5 120 dilution), and negative control (PBS-T) was added. The plates were then incubated for 2 hr at 37°C. After four washes with PBS-T, 100 µl of HRP-conjugated anti-CHIKV HIS or mAb 152 diluted 1:10 000 in PBS-T with 2.5% of normal bovine serum was added. The plates were incubated for 1 hr at 37°C and washed five times with PBS-T. The HRP-catalyzed reaction was carried out as described for the MAC-ELISA methods. The percentage inhibition (%I) was calculated using the following formula: %I = (1-[sample OD/mean OD of negative controls]) x 100%. Sample titer was then calculated using the Reed and Muench method , accordiTiter = exponential [(%I of the lowest sample dilution with a %I ? 50% inhibition - %I of the highest sample dilution with a %I < 50%) / (%I of the lowest sample dilution with a %≥ 50% inhibition – 50) + log of the lowest sample dilution with a %I ? 50% inhibition]Sample size was calculated using Epidat® software version 4.1  and the following parameters: precision of the sensitivity and specificity estimates, 5%; expected sensitivity and specificity, 95%; and confidence level, 95%. The calculated sample size was at least 193 samples. The sensitivity, specificity, positive and negative predictive values, and their 95% Confidence Intervals (95% CI), as well as kappa indices, were calculated using the statistical package Stata®/MP13.1 .First, the optimal concentrations of the major components of the four serological tests were established. Optimal concentrations of MAC-ELISA-HIS and MAC-ELISA-mAb were determined using a saturation curve . OptimalA summary of the protocols for the two newly developed, in-house MAC-ELISA systems compared to the CDC-MAC-ELISA  is shownA total of 198 serum samples collected from suspected chikungunya cases with known CDC-MAC-ELISA results were processed using the two in-house MAC- ELISA systems and compared to the CDC method. Both in-house systems yielded sensitivity, specificity, and predictive values above 90%  when usiA total of 260 paired acuteand convalescent-phase serum samples from suspected chikungunya cases enrolled in one of two ongoing studies of dengue and chikungunya conducted in Managua, Nicaragua , 18, werSensitivity, specificity, and positive and negative predictive values for all four serological methods were calculated . The fouThis study reports the development, standardization, and clinical evaluation of two MAC-ELISAs and two IEMs for the diagnosis and surveillance of chikungunya. In order to evaluate our MAC-ELISA systems, 198 serum samples collected via the national surveillance system were processed using both (MAC-ELISA-HIS and the MAC-ELISA-mAb) ELISA and compared to the CDC-MAC-ELISA as the reference method. A sensitivity of 97.4% and 96.6% and a specificity of 98.8% and 91.5% were obtained for the MAC-ELISA-HIS and MAC-ELISA-mAb, respectively. These results are consistent with results reported for similar assays. For instance, the MAC-ELISA developed by Wasonga and colleagues had a sensitivity of 97.6% and a specificity of 81.3% compared to the CDC-MAC-ELISA. Blacksel and colleagues  evaluateA substantial achievement of the present study was to reduce the processing time from 48 hours for the CDC-MAC-ELISA to 2.5 – 3 hours for the in-house MAC-ELISA systems. In addition, the in-house MAC-ELISA assays enabled savings in supplies and reagents because they do not use a non-viral antigen control for each sample. Thus, 90 samples can be processed in one plate without affecting the assay specificity, whereas the CDC technique only allows 45 samples per plate. The decision to use more concentrated samples (1 : 20 instead of 1 : 400 as recommended in the CDC protocol) was made to facilitate the work-flow. The lower dilution allows the samples to be diluted directly into the plate, instead of having to predilute the samples prior to adding them to the plate.In addition to the assays for detecting CHIKV-specific IgM antibodies, we developed two Inhibition ELISA methods that measure total anti-CHIKV antibodies. These assays can be used for diagnosis of chikungunya in paired acute and convalescent samples. In addition, they can be used to conduct seroprevalence studies that enable determination of current chikungunya epidemic’s magnitude, and therefore, help predict the potential of future epidemics. A similar method for the detection of total anti-dengue virus antibodies has been used in Nicaragua for 17 years by a clinical study of dengue at the National Pediatric Reference Hospital  and for 12 years in the Pediatric Dengue Cohort Study , 28. BasA total of 260 paired acute and convalescent samples collected through both Nicaraguan research studies were processed using all four new serological methods. Results were compared to those from a CHIKV rRT–PCR assay performed on the acute sample. As expected, the sensitivity of the MAC-ELISA was low for samples collected in the acute phase (average of 2.0 days after symptoms onset), but high for samples collected in the convalescent phase (average of 21.6 days after symptom onset). This result was consistent with the fact that detection of IgM is not useful in the first days of illness  – 32. ThOne limitation of the study was the lack of a gold standard method for the standardization of the IEM. However, the clinical evaluation of the IEM with the 260 paired samples demonstrated the utility of this technique, with ~95% sensitivity and specificity when compared with testing of acute samples by rRT-PCR. These results are similar to those obtained by Balmaseda and colleagues  when theSince the standardization and evaluation of these different serological methods was accomplished, the chikungunya MAC-ELISA-HIS assay has been decentralized to 12 regional laboratories in the country, a significant improvement for national epidemiological surveillance. Currently, all of these laboratories are able to identify and differentiate dengue and chikungunya cases using MAC-ELISA. Also, two studies, one local and one national, of chikungunya seroprevalence were conducted using the IEM assays to determine the attack rate of CHIKV infection in Nicaragua .Two MAC-ELISAs and two IEMs for chikungunya diagnosis and research were developed, standardized, and evaluated by this study. These assays currently support epidemiological surveillance of chikungunya at the national level in Nicaragua, as well as more focused research studies in Managua. Nicaragua has thus become the first country in Central America with the capacity to produce its own reagents for serological diagnosis of chikungunya. Nicaragua’s experience can be extended to other countries, and thus contribute to the sustainability of chikungunya diagnosis and to the fight against the dengue and chikungunya epidemics that are currently affecting the Region of the Americas.We are grateful to Michael S. Diamond at Washington University  for providing the CHIKV mAbs and 181/25 vaccine strain. We also thank José Luis Soto for his significant contribution in the production of hyperimmune serum; María José Vargas, Stephania Ramírez, and Damaris Collado for their help in processing the samples; Andrea Núñez for her invaluable help in the selection of samples and Douglas Elizondo for his significant help on formatting and editing the text. This work was supported by a supplement to R01 AI099631 (AB) from the United States National Institutes of Health .RPSP/ PAJPH and/or PAHO.Authors hold sole responsibility for the views expressed in the manuscript, which may not necessarily reflect the opinion or policy of the


Objectives. To assess visually color-matching and blending-effect (BE) of a universal shade bulk-fill-resin-composite placed in resin-composite-models with different shades and cavity sizes and in natural teeth (extracted and patients' teeth). Materials and Methods. Resin-composite-discs (10 mm × 1 mm) were prepared of universal shade composite and resin-composite of shades: A1, A2, A3, A3.5, and A4. Spectrophotometric-color-measurement was performed to calculate color-difference (ΔE) between the universal shade and shaded-resin-composites discs and determine their translucency-parameter (TP). Visual assessment was performed by seven normal-color-vision-observers to determine the color-matching between the universal shade and each shade, under Illuminant D65. Color-matching visual scoring (VS) values were expressed numerically (1–5): 1: mismatch/totally unacceptable, 2: Poor-Match/hardly acceptable, 3: Good-Match/acceptable, 4: Close-Match/small-difference, and 5: Exact-Match/no-color-difference. Occlusal cavities of different sizes were prepared in teeth-like resin-composite-models with shades A1, A2, A3, A3.5, and A4. The cavities were filled by the universal shade composite. The same scale was used to score color-matching between the fillings and composite-models. BE was calculated as difference in mean-visual-scores in models and that of discs. Extracted teeth with two different class I-cavity sizes as well as ten patients' lower posterior molars with occlusal caries were prepared, filled by universal shade composite, and assessed similarly. Results. In models, the universal shade composite showed close matching in the different cavity sizes and surrounding shades (4 ≤ VS < 5) . In extracted teeth, there was good-to-close color-matching  . In patients' molars, the universal shade composite showed good-matching . Conclusions. Color-matching of universal shade resin-composite was satisfactory rather than perfect in patients' teeth. Blending-effect (BE) of a restorative dental material refers to its ability to acquire a color resembling that of the adjacent structure . Some maA main previous research by Paravina et al.  investigThe blending-effect of layered composite was evaluated in another study by the same authors . The preUniversal shade bulk-fill composite, in addition to its increased curing depth, is claimed by the manufacturer to have chameleon or BE. There is no published research on the blending capacity of this universal shade composite. The purpose of this study was to assess the color-matching and the BE of the universal shade composite in different restoration sizes and surrounding shades in resin-composite-models and natural teeth (extracted and patients' teeth).The following materials were used in this study: universal shade bulk-fill resin-composite and five composite shades  . The pren = 6/shade), Thirty-six discs (10 mm × 1 mm) were prepared from the universal shade and shaded resin-composites 2 + (Δa∗)2 + (Δb∗)2]1/2, where L∗, a∗, and b∗ are the CIELAB color coordinates [The color parameters of the discs were measured using a spectrophotometer . The 10° observer function was used with the CIE Illuminant D65. Δrdinates .LW∗ − LB∗)2 + (aW∗ − aB∗)2 + (bW∗ − bB∗)2]1/2, where L∗, a∗, and b∗ are the CIELAB color coordinates, while W and B refer to the values for each specimen against the white and black backgrounds, respectively [L∗ = 11.24, a∗ = 0.17, and b∗ = 0.28) and the white standard tile  were used as the standard backgrounds for the specimens during the color measurements. The results of ΔE and TP were analyzed by one-way ANOVA followed by Bonferroni's post hoc test to detect significant differences in values (P ≤ 0.05). Statistical analysis was performed with IBM® SPSS® Statistics Version 20 for Windows.The TP of the discs was calculated as the color-difference between specimens placed against white and black backgrounds , 6. A hiectively . The blaVisual assessment was performed by seven female observers (four dentists and three scientists), with normal-color-vision as checked by Ishiara test. To determine the color-matching between the universal shade and each shaded composite disc, color-matching visual scoring (VS) values were expressed numerically as follows: 1: mismatch/totally unacceptable, 2: Poor-Match/hardly acceptable, 3: Good-Match/acceptable, 4: Close-Match/small-difference, and 5: Exact-Match/no-color-difference. All observers were trained in color-matching and taught about the used grading system before the assessment.A color assessment cabinet , with neutral grey walls and floor, was used for the visual evaluation. Illuminant D65 was selected. The cabinet had an oblique grey specimens' holder providing a 45° angle between the specimens and the illuminant. The observers placed their head on a head holder allowing an observation distance of 25 cm from the specimens and looking perpendicularly upon them. Thus, the illuminant/viewing geometry was 45°/normal. During assessments, external lights were turned off and, after an adaptation period, the observers scored the color-matching between the universal shade and shaded composite discs which were in edge contact. Results were recorded and mean values were calculated. Inter- and intraobserver agreements were also recorded.The interobserver agreement was calculated as the mean value of the highest percentage of observers that graded specimens of each shade identically . The intraobserver agreement was calculated as the mean value of the highest percentage of identical scores for the six specimens of the same shade by a specific observer .The dimensions of the models and the prepared cavities are shown in Class I occlusal cavities were prepared in each acrylic tooth, one with a small cavity size , Rubber base impressions were taken for the coronal portions of the acrylic teeth after the cavities being prepared, To construct each resin-composite-model, the previous impression was filled with a shaded resin-composite, The models with the identical prepared cavities were restored by the universal shade bulk-fill resin-composite, The color-matching VS for the fillings versus the different shades of models with different cavity sizes was assessed as before. BE was calculated as a difference in color-matching scores for the models versus that for composite disc of the same shade (BE = VS of models − VS of separate discs). The interobserver and intraobserver agreements were calculated as before.Freshly extracted human lower molars sound natural teeth were selected for this study. The shades of the teeth were assessed by a spectrophotometer . Two different class I-cavity sizes were prepared as before, filled by universal shade composite, Ten patients with occlusal caries in lower posterior molars were selected to participate in this study, after getting the approval of the research ethics committee and the patients' informed consents. The shades of their teeth were determined using VITA Easyshade spectrophotometer. After class I cavities had been prepared, a 37% phosphoric acid etching gel  was applied for 15 seconds, then rinsed, and air-dried. A bonding agent  was applied to the teeth in a rubbing motion for 20 seconds. Then, a gentle stream of air was applied over the bonding agent for 5 seconds and then it was light-cured for 10 seconds. The universal shade bulk-fill composites  were used to fill the cavities and then light-cured for 20 seconds. Photographs of some representative clinical cases are shown in Figures 2 room with ceiling height of 2.82 m and eliminated with color corrected florescent lambs). The walls of the clinic were painted with an off-white color .Visual assessment for the filling color-matching was performed by the same observers using the same scoring system. The assessment was performed in a clinic related to the dental school  in upright position. The light source of the dental unit  was focused on the patients' mouth from the distance of one meter. Then, each patient was asked to open his mouth and the observers assessed the color-matching of the filled tooth. The observers performed the evaluation twice for the color-matching of the universal shade composite filling. The BE and interobserver and intraobserver agreements were calculated as before.E values of the universal shade versus the shaded composite discs and their TP are presented in E increased as the shades become darker. The universal shade showed the highest TP (26.7 ± 0.7).ΔThe VS values of the universal shade composite versus the shaded composite discs and models are shown in Since BE is calculated by subtracting VS of separate discs from that in models, thus, BE values for shade A1 in small and large cavities were 0.6 (= 4.8 subtracted by 4.2) and 0.5 (= 4.7 subtracted by 4.2), respectively. Meanwhile BE of shade A4 were 2.9 (= 4.1 subtracted by 1.2) and 2.8 (= 4 subtracted by 1.2), respectively. The BE values in composite-models increased with darker shades and increasing the color-difference between the filling and surrounding shade separately.The shades of the extracted teeth were A4 as measured by the VITA Easyshade spectrophotometer. The interobserver agreement was 82.2% for single composite discs and 75.9% and 82.2% for models with small and large cavities, respectively. Meanwhile interobserver agreements for extracted teeth with small and large cavities were 79.3% and 89.4%, respectively. In the patients' teeth, the interobserver agreement was 92.5%.However, the intraobserver agreements for single composite discs and models with small and large cavities were 86.6%, 84.3%, and 87.3%, respectively. Meanwhile intraobserver agreements for extracted teeth with small and large cavities and the patients' teeth were 86%, 91.9%, and 93.2%, respectively.Universal shade bulk fill resin-composite may represent a smart solution in color-matching if it blends with the adjacent tooth structure shade, beside its increased curing depth. This material is indicated for posterior fillings (classes I and II), not in anterior teeth (classes III and IV). In the latter case, the high translucency of this resin-composite would transmit the background color of the oral cavity.In this study, resin-composite-models were constructed to evaluate the BE, rather than disc-shaped specimens, in an attempt to resemble the natural teeth and restorations in morphology and dimensions. In addition, these models standardized the sizes of the cavities and restorations as well as the surrounding wall thicknesses and shades. However, discs were used for spectrophotometric measurements as they had flat surface for accurate results.The BE in this study were determined by the difference in mean scores  as it was shown previously in another research that the computation of mean category values was valid and that differences between mean category values can be used to represent the blending-effect .E values. Although there are several formulas for color-difference calculation, the most commonly used one in dental research is obtained from the CIE-L∗a∗b∗ system [The universal shade composite fillings showed close color-matching in the composite-models despite their great color variation , as verified by Δ∗ system .The high color-matching of the universal shade composite might be attributed to its high translucency reflecting the shade of the surrounding walls, even with different shades and translucency. This was in agreement with Paravina et al.  who concIt was noticed that the BE of the universal shade composite in darker models was higher as numerical values, compared to that in lighter one. Yet, its color-matching was higher in lighter shades. In our study, the BE values increased with increasing the color-difference between the universal shade and the surrounding composites. This was contradictory to what was concluded by Paravina et al.  that theComparing the BE among the different cavity sizes within the same model shades, it was shown that the BE value decreased when the size of the cavity increased. This was in agreement with Paravina et al.  who concθ) calculated as follows: 2θ = arctan(r/d), where r is the radius of a specimen and d is the distance from the observer [It was stated by Paravina et al.  that “thobserver , 16.The BE of the extracted teeth differed from that of the models. This may be due to the difference in their nature; for example, natural teeth are multilayered and polychromatic . The extThe inter- and intraobserver agreement in this study were relatively high as those observed in Paravina et al.  and contThe universal shade bulk-fill resin-composite showed acceptable color-matching in vivo; however it may not be the optimal selection when esthetics is the patient's prime concern. It may be better to assess color-matching and blending-effect in vivo rather than in vitro as it is a better simulation of clinical condition.


Only a small number of patients survive out-of-hospital-cardiac-arrest (OHCA). The duration of CPR varies considerably and transportation of patients under CPR is often unsuccessful. Termination-of-resuscitation (TOR)-criteria aim to preclude futile resuscitation efforts.Our goal was to find out to which extent existing TOR-criteria can be transferred to paediatric OHCA-patients with special regard to their prognostic value.We performed a retrospective analysis of an eleven-year single centre patient cohort. 43 paediatric patients admitted to our institution after emergency-medical-system (EMS)-confirmed OHCA from 2003 to 2013 were included. Morrison’s BLS- and ALS-TOR-rules as well as the Trauma-TOR-criteria by the American Association of EMS Physicians were evaluated for application in children, by calculating sensitivity, specificity, negative and positive predictive value for death-, as well as survival-prediction in our cohort.n = 7 PCPC 1/2, n = 7 PCPC 5). Sensitivity for BLS-TOR-criteria predicting death was 48.3%, specificity 92.9%, the PPV 93.3% and the NPV 46.4%. ALS-TOR-criteria for death had a sensitivity of 10.3%, specificity of 100%, a PPV of 100% and an NPV of 35%.26 patients achieved ROSC and 14 were discharged alive (Retrospective application of the BLS-TOR-rule in our patient cohort identified the resuscitation of one later survivor as futile. ALS-TOR-criteria did not give false predictions of death. The proportion of CPRs that could have been abandoned is 48.2% for the BLS-TOR and only 10.3% for the ALS-TOR-rule. Both rules therefore appear not to be transferable to a paediatric population. The incidence for paediatric out-of-hospital-cardiac-arrests (OHCA) is rather uniform throughout developed countries with 7.3-9.1-cases per 100 000 person-years .Survival rates for paediatric and adult OHCAs are equally poor. Two to ten percent of the children who are resuscitated in the prehospital setting survive. 50% of these without neurological deficits , 7.Prospects of survival are determined by the duration of untreated cardiopulmonary arrest, witnessing, potentially reversible causes, duration of cardiopulmonary resuscitation (CPR), return-of-spontaneous circulation (ROSC), heart rhythm, blood-pH, therapy and the perceptibility of definite life-extinction –11. TermTOR-criteria have been established in the treatment of traumatic-cardiopulmonary-arrests (TCPA) in about half of US-states. The 2015 European Resuscitation Council (ERC)-Guidelines strongly advocate for their use , 12. TheValidated criteria exist only for adult OHCAs because children are routinely excluded from TOR-protocols , 16–18. One reason for the reluctance to use TOR-rules in children, is a lack of empirical data regarding the safe transferability: only one study has examined the eligibility of TOR-criteria for children . TherefoOur goal was to find out, to which extent TOR-criteria can be transferred to paediatric OHCA-patients with special regard to prognostic validity.The study was approved by the local ethics committee of our institution (case number 173/14). The Institutional Review Board waived the need for informed consent as the study is of a retrospective design.We performed a retrospective analysis of an eleven-year single centre patient cohort.All consecutive patients younger than 18 years admitted to our institution after out-of-hospital- resuscitation (OHR) between 1st January 2003 and 1st January 2014 were tested for eligibility.Included were patients with cardiac arrest confirmed by emergency-medical-system (EMS)-personnel. Excluded were children with respiratory arrest only or ROSC prior to EMS-arrival. Patients who were pronounced dead in the pre-hospital setting could not be included into this study since they are not admitted to our institution and data were not accessible.We examined established adult TOR-rules under the hypothesis that they would have been applied during the CPR of the patients who are included. Primary endpoint was the  prediction of death in later survivors. Secondary endpoints were the neurocognitive outcome in these survivors measured as Paediatric Cerebral Performance Catetgory (PCPC) and the proportion of futile resuscitations in which TOR-criteria would have predicted death. Characteristics of OHCA and resuscitations were recorded and are presented in accordance with Utstein reporting rules .The Paediatric Cerebral Performance Category (PCPC) by Fiser graduates neurological function into 6 categories: PCPC 1 represents a normal neurological function, PCPC 6 a braindead patient. PCPC 2 is a mild disability with a minor neurological handicap that does not interfere with daily activity. Moderate disability is a PCPC 3  and PCPC 4 is a severe disability (extensive dependence on others for daily activities and unable to attend school). PCPC 5 represents a patient who is in a vegetative state (VS), defined as any degree of coma without fulfilling the criteria for brain death. Criteria include no interaction with the environment even if awake, no cerebral responsiveness or evidence of cortical function and no reaction to verbal stimuli .RWTH Aachen University Hospital is an academic tertiary care centre. 225000 patients are treated every year. 250000 inhabitants live in the catchment area of 160skm. 35000 of these are under 18 years.German EMS regularly provide teams with one trained emergency physician and at least two paramedics for severe preclinical emergencies. The prevailing concept in the German EMS is “Treat and Run”, meaning the immediate stabilisation of vital parameters on scene to secure the patient’s transportability. For Aachen and the federal state of Nordrhein-Westfalen the legally enforced arrival time is eight minutes within municipalities and twelve minutes within rural areas.Since our institution is the only tertiary care centre in the region covering the full spectrum of traumatology including head injuries and burns as well as having a catchment area that is almost equally urban and rural, study settings were representative for the population of interest.Criteria for an early identification of hopeless cases in preclinical resuscitation have to be kept simple and ought to be absolutely reliable. Understanding these criteria as a statistical test, the test’s perception on prediction of death or survival determines the statistical figures for reliability: If death is predicted, specificity and positive predictive value (PPV) both need to equal 100%. PPV states how many patients in whom the rule would advise TOR (corresponding to a positive test) will actually die.If the test predicts survival, sensitivity and negative predictive value (NPV) both need to equal 100%.Verbeek et al. published variables that were retrospectively predictive for death .For prospective validation of Verbeek’s variables, Morrison et al. published a study about TOR during basic-life-support (BLS).According to the BLS-TOR rule, resuscitation can be terminated when all three following negative predictors are present: no defibrillation performed, no ROSC before transport and arrest not witnessed by EMS-personnel. Morrison et al. found a specificity of 90.2% (95% CI 88.4-91.8) and a PPV for death of 99.5%. Survival was reported for 0.5% of the patients, in whom the rule would have allowed TOR. Sensitivity for death was 64.4% (95% CI 61.6-67) .witnessed by bystanderswitnessed by EMS-personnelbystander-CPR performedshock deliveredROSC before transportIn 2007 Morrison et al. extended the BLS-TOR-criteria to generate TOR-criteria for advanced-life-support (ALS). According to the ALS-TOR-rule, a resuscitation may be terminated when none of the five following positive criteria for survival is met:Sensitivity and NPV for survival were 100% (95% CI 99.9-100).According to the ALS-TOR-rule 32% (95% CI 30–33) of the patients were to be pronounced dead on scene. The PPV for survival was 7% (95% CI 6.6-8.1).Applying the BLS-TOR-rule on the same cohort of patients, sensitivity and NPV for survival both equal 100% (95% CI 99.9-100) and 48% of the patients could have been pronounced dead on scene.Other studies confirmed the reliability on various cohorts , 22–25.BLS-criteria find their statistical correspondence for security in the PPV. ALS-criteria on the other hand, focus on absence of positive predictors, so that NPV is the decisive parameter.In the US there are very few studies analysing TOR-criteria on paediatric patients because children are usually not included into TOR-protocols , 18.Capizzani et al. , analyseData were collected from EMS and PICU charts (electronic and paper-bound) and part of the data for patients of the years 2011 to 2013 from the German Resuscitation Registry. Missing data were excluded from statistical analysis.Chart abstraction was performed systematically and Utstein-data were directly transmitted from paper charts to spreadsheets . Data coThe data collector was trained in Utstein-style reporting so consistency in definitions is guaranteed.The criterion of “witnessing” was defined uniformly when the child was not alone during arrest and “bystander-CPR” as any effort to restore circulation.We calculated sensitivity, specificity, NPV and PPV for death-, as well as survival-prediction on our cohort. Data collection was in accordance with Utstein-guidelines . Calculan = 6 (14%), PCPC 2: n = 1 (2.3%), PCPC 5: n = 7 (16.3%)) and 12 died on the PICU (27.9%). None of the patients had an advance directive or do not attempt resuscitation (DNAR) code prior to the event. The average age of our patients was 6.03 years. 21 patients (48.8%) were male. 15 had relevant comorbidity (34.9%). Affected were seven survivors, among these one child with a complex congenital heart defect , one child with multiple chromosomal defects, one former gemini-preterm infant and one former preterm infant with a congenital heart defect , one patient with hypertophic cardiomyopathy, one child with epilepsy and one with mental retardation of unknown origin.Of 75 paediatric patients with OHR and consecutive hospital admission, 43 cases with cardiac arrest confirmed by emergency-medical-system (EMS)-personnel were included. 32 patients were excluded, 19 because of respiratory arrest without cardiac arrest, 13 because of ROSC prior to EMS-arrival. Of the 43 patients with EMS-confirmed cardiac arrest 17 patients were admitted under ongoing CPR, 26 after ROSC .Main causes for CPR were trauma and other non-cardiac causes, such as drowning, sudden infant death syndrome (SIDS)/ acute life threatening event (ALTE) and epilepsy.Fifteen patients met all three BLS-TOR-criteria. One among these, however, survived.Therefore, sensitivity for the prognosis of death was 48.3% , specificity 92.9% , PPV 93.3% .The odds ratio was 12.1 (95% CI 1.4-105.3)  met all five ALS-TOR-criteria so that termination-of-resuscitation could have been considered. The sensitivity was 10.3% , specificity 100% , PPV 100%  and NPV 35%  in our cohort.None of the 14 surviving children met all the criteria for survival.However, 40 patients met at least one of the criteria associated with survival Fig. .Fig. 3Adn = 10) and 37.9 ± 28.8 min in patients who died (duration known for n = 16).We compared Capizzani’s rules to our own cohort even though they are only intended for trauma patients, while only twelve of our patients had a traumatic cardiac arrest. We tested how many patients received CPR for more than 15 min and had fixed pupils upon EMS arrival. Mean CPR-duration was 19.3 ± 19.5 min in survivors , specificity 90.9% (CI 95% 73.9-107.9), PPV 94.4% (CI 95% 83.9- 105) and NPV 66.7% (CI 95% 26.3-69).While validated TOR-criteria exist for adult OHCAs, they have not yet been established for the paediatric population , 16–18. The Morrison-BLS-TOR-rule is explicitly mentioned in the 2015 ERC-Guideline on adult ALS and is the most commonly used and best validated TOR-rule , 22–25. Morrison’s ALS-TOR-criteria were 100% specific in our cohort and had a 100% PPV. Thus, no resuscitation would have been classified as futile in any of the survivors. With a sensitivity of 10.34% the rule would have only been applicable in one tenth of the patients who later died.One reason for ALS-TOR-rule’s limited sensitivity in the paediatric population is the different aetiology of cardiac arrest and presenting rhythm in children. Morrison et al. included only OHR due to a cardiac cause. Leading aetiologies in paediatric OHCA are trauma and SIDS . DefibriFor a legally qualified pronouncement of death “in the field” without obvious signs of definite death, the presence of a physician is obligatory. In the Anglo-American system, patients without recognition of life extinct (ROLE) are routinely transported to a hospital, where the decision to continue or withdraw CPR is made by a physician. This proceeding leads to a high proportion of futile transportations and has triggered the demand for an “exception-rule” to systematically and safely identify OHCA-patients without any prospect of survival.In Germany adults are less frequently transported with ongoing CPR, due to the common presence of a physician on scene. However, our impression is that emergency physicians tend to transport a higher proportion of children than adults under ongoing CPR, although published data are lacking.n = 1: PCPC 1, n = 1: PCPC 5).It is therefore remarkable that we found two surviving children who were transported with ongoing CPR (Trauma-TOR-criteria failed to reach 100% in specificity (90.9%) and PPV (94.4%) for survival. However, the one survivor had PCPC 5.In the “American College of Surgeons Committee on Trauma” joint-position-statement CPR-duration was found to be the most important predictor for outcome, showing enormous variability at the same time. The statement recommends to initiate a decision-making between family and professionals about terminating the resuscitation if there is no ROSC after 30 min and the next hospital is not reachable within 30 min . UniformThe central benefit of TOR-criteria is to preclude “futile” resuscitations. When the arrest happens at home or is witnessed by family members, continuing a futile resuscitation can interfere with relatives’ ability to make their farewells. This is most likely to be unfavourable for long-term coping, although no systematic research for family needs in paediatric OHCAs was performed yet . Intervi“Futility” can be doubtlessly assumed for those cases without a prospect of survival. We chose this narrowest definition of futility for our conclusions from this study. Many publications use a broader concept of futility that considers the functional quality of life in case of survival though. The 2010 ERC-Guideline states “Futility exists if resuscitation will be of no benefit in terms of prolonging life of acceptable quality”. The 2015-Guideline now adopted the more cautious WMA-definition . It defiEmergency personnel report a reluctance to stop paediatric resuscitations because they fear the parents’ reactions . TOR-criAnother argument in favour of TOR-criteria is a reduction in accident hazard because emergency transportations using warning devices are reduced.They can also save resources that are valuable to other, less hopeless patients, ideally without restricting the prospects of the patient concerned.In summary, TOR-rules as a standardized approach are as desirable for children as for adults.Neither Morrison’s BLS-TOR-rule, nor the TOR-rule for TPCA were absolutely predictive for death in our cohort. The one survivor whose CPR would have been classified as futile by both rules, had a poor neurological outcome (PCPC 5). The ALS-TOR-rule reached 100% for specificity and PPV, but could have been applied to only three of the 29 deceased patients.Although the vast majority of transportations under ongoing CPR are futile , 29, thiThe current study is limited by its small cohort and retrospective design as some data are therefore missing. The overall outcome of patients is better than reported , the likely reason being that we could only include those patients who were actually admitted to the Emergency Department and PICU of our institution, but not those who were declared dead on scene. This aspect is also important for the interpretation of the statistics. It is likely, that we overestimate the proportion of survivors. Apart from that, results may also be also influenced by the fact that all causes of cardiac arrest were included because some cardiac arrest causes are more easily reversible than others.